In order to generate the antigen receptor repertoire that mediates mammalian immunity, lymphocytes must assemble immunoglobulin (Ig) and T cell receptor (TCR) genes from separate coding segments via V(D)J recombination. Importantly, V(D)J recombination must be tightly regulated to ensure proper lymphocyte development and to avoid chromosomal translocations that cause lymphoid tumors. Chromatin accessibility of V, D, and J segments is governed by tissue/stage-specific enhancers, which affect proper targeting of V(D)J recombinase. The applicant's laboratory has recently discovered that the germline promoters flanking unrearranged gene segments are also required for recombinase accessibility. The TCRbeta germline promoter regulates accessibility via a spatially restricted mechanism that operates independent of transcription through target gene segments. Coupled with prior enhancer studies, these two fundamental discoveries highlight a new role for the germline promoter as a second accessibility control element (ACE) in the process of antigen receptor gene assembly. We hypothesize that ACEs mediate a series of biochemical events that culminate in a recombinase-accessible locus, including (i) enhancer-directed opening of local chromatin, (ii) formation of a higher order enhancer/promoter complex (i.e., an enhanceosome), (iii) activation of the promoter, and (iv) chromatin remodeling of promoter-proximal nucleosomes. To test this central hypothesis, we will define the nucleosome remodeling events under ACE control that yield a recombinase-accessible locus (Specific Aim 1). In Specific Aim 2, we will identify the chromatin modifiers that ACEs recruit to establish the requisite histone code for remodeling. Aim 3 studies will reveal the specific transcription factors that facilitate ACE cross-talk and contribute to the formation of a locus poised for rearrangement. Overall, the project will establish the molecular mechanisms by which V(D)J recombinase selects specific targets from an extremely large array of gene segments, resulting in a fully functional immune repertoire. The proposed research will also provide fundamental insights into the processes that control chromatin accessibility during selective transcription and replication of genomic information in all eukaryotic cells.